The present invention relates to atheromatous plaque associated molecules for prevention and treatment of atherosclerosis and related disease and, more particularly, to methods and compositions employing plaque associated molecules effective in inducing mucosal tolerance and inhibiting inflammatory processes contributing to atheromatous vascular disease and sequalae.
Cardiovascular disease is a major health risk throughout the industrialized world. Atherosclerosis, the most prevalent of cardiovascular diseases, is the principal cause of heart attack, stroke, and gangrene of the extremities, and as such, the principal cause of death in the United States. Atherosclerosis is a complex disease involving many cell types and molecular factors (for a detailed review, see Ross, 1993, Nature 362: 801-809). The process, which occurs in response to insults to the endothelium and smooth muscle cells (SMCs) of the wall of the artery, consists of the formation of fibrofatty and fibrous lesions or plaques, preceded and accompanied by inflammation. The advanced lesions of atherosclerosis may occlude the artery concerned, and result from an excessive inflammatory-fibroproliferative response to numerous different forms of insult. For example, shear stresses are thought to be responsible for the frequent occurrence of atherosclerotic plaques in regions of the circulatory system where turbulent blood flow occurs, such as branch points and irregular structures.
The first observable event in the formation of an atherosclerotic plaque occurs when inflammatory cells such as monocyte-derived macrophages adhere to the vascular endothelial layer and transmigrate through to the sub-endothelial space. Elevated plasma LDL levels lead to lipid engorgement of the vessel walls, with adjacent endothelial cells producing oxidized low density lipoprotein (LDL). In addition, lipoprotein entrapment by the extracellular matrix leads to progressive oxidation of LDL by lipoxygenases, reactive oxygen species, peroxynitrite and/or myeloperoxidase as well as other oxidizing compounds. These oxidized forms of LDLs are then taken up in large amounts by vascular cells through scavenger receptors expressed on their surfaces.
Lipid-filled monocytes and smooth-muscle derived cells are called foam cells, and are the major constituent of the fatty streak. Interactions between foam cells and the endothelial and smooth muscle cells surrounding them produce a state of chronic local inflammation which can eventually lead to activation of endothelial cells, increased macrophage apoptosis, smooth muscle cell proliferation and migration, and the formation of a fibrous plaque (Hajjar, D P and Haberland, M E, J. Biol Chem 1997 Sep. 12; 272(37):22975-78). Such plaques occlude the blood vessels concerned and thus restrict the flow of blood, resulting in ischemia, a condition characterized by a lack of oxygen supply in tissues of organs due to inadequate perfusion. When the involved arteries block the blood flow to the heart, a person is afflicted with a ‘heart attack’; when the brain arteries occlude, the person experiences a stroke. When arteries to the limbs narrow, the result is severe pain, decreased physical mobility and possibly the need for amputation.
Oxidized LDL has been implicated in the pathogenesis of atherosclerosis and atherothrombosis, by it's action on monocytes and smooth muscle cells, and by inducing endothelial cell apoptosis, impairing anticoagulant balance in the endothelium. Oxidized LDL also inhibits anti-atherogenic HDL-associated breakdown of oxidized phospholipids (Mertens, A and Holvoet, P, FASEB J 2001 October; 15(12):2073-84). This association is also supported by many studies demonstrating the presence of oxidized LDL in the plaques in various animal models of atherogenesis; the retardation of atherogenesis through inhibition of oxidation by pharmacological and/or genetic manipulations; and the promising results of some of the interventionaltrials with anti-oxidant vitamins (see, for example, Witztum J and Steinberg, D, Trends Cardiovasc Med 2001 April-May; 11(3-4):93-102 for a review of current literature). Indeed, oxidized LDL and malondialdehyde (MDA)-modified LDL have been recently proposed as accurate blood markers for 1st and 2nd stages of coronary artery disease (U.S. Pat. Nos. 6,309,888 to Holvoet et al and 6,255,070 to Witztum, et al).
Reduction of LDL oxidation and activity has been the target of a number of suggested clinical applications for treatment and prevention of cardiovascular disease. Bucala, et al (U.S. Pat. No. 5,869,534) discloses methods for the modulation of lipid peroxidation by reducing advanced glycosylation end product, lipid characteristic of age-, disease- and diabetes-related foam cell formation. Tang et al, at Incyte Pharmaceuticals, Inc. (U.S. Pat. No. 5,945,308) have disclosed the identification and proposed clinical application of a Human Oxidized LDL Receptor in the treatment of cardiovascular and autoimmune diseases and cancer.
Another abundant atherogenesis-related plaque component is Beta 2-Glycoprotein I. Beta2-Glycoprotein I (Beta2GPI) is a 50-kDa molecule that acts as an anticoagulant in in-vitro assays. Although the exact role of Beta2GPI in atherogenesis has yet to be elucidated, several relevant properties have been observed: 1) it is able to bind negatively charged phospholipids or phospholipid-expressing cells (apoptotic cells, activated platelets); 2) it is able to bind to modified cellular surfaces, enhancing their clearance by scavenging macrophages (Chonn A, et al J Biol Chem 1995; 270: 25845-49; and Thiagarajan P, et al Arterioscler Thromb Vasc Biol 1999; 19:2807-11); and 3) it is an important target for binding of autoimmune antiphospholipid antibodies (aPLs). Beta2GPI has to undergo structural alteration in order to be recognized by aPLs. This alteration may be initiated, for example, by binding to negatively charged phospholipids or high binding plates, but also in vivo by binding apoptotic cells that express phophatidylserine.
Recent studies investigating the importance of anti Beta2GPI antibodies in promoting a procoagulant state have focused on the effects of these antibodies on cellular and protein components of the coagulation system (endothelial cells, platelets and macrophages; tissue factor and coagulation factors). These studies indicate that anti Beta2GPI antibodies prevent the deactivation of platelets, sustaining their phagocytic clearance; interact with late endosomes of human endothelial cells; and suppress the inhibitory activity of the tissue factor pathway inhibitor. This association with coagulation events is consistent with Beta2GPIs proposed function in the prothrombotic antiphospholipid syndrome (APLS). Both U.S. Pat. Nos. 5,998,223 and 5,344,758 (to Matsuura, et al and Krilis, et al, respectively) disclose the application of anti Beta2GPI antibodies, some to cryptic epitopes, for diagnostics in APLS and SLE. However, no therapeutic applications are disclosed by the authors.
A third important plaque-related component associated with atherogenesis is the 60/65 kDa heat shock protein (HSP). This mitochondrial protein is a member of the HSP family, which constitutes nearly 24 proteins displaying high degree of sequence homologies between different species. These proteins, as their name implies, are expressed in response to stresses including exposure to free radicals, heat, mechanical shear stress, infections and cytokines, and protect against unfolding and denaturation of cellular proteins. This has led to their designation as molecular ‘chaperones’. However, HSP function may have undesired consequences, since over expression of HSPs may, under certain conditions promote an autoimmune reaction with resultant tissue damage. The mechanisms responsible for the HSP immune mediated damage are as yet unclear: it is presumed that cryptic, “non-self” neo-epitopes are exposed following their upregulation. Alternatively, it was suggested that cross-reaction exists between self-HSP and ‘foreign’ HSP epitopes introduced following infections which may trigger a pathological, autoimmune response against native HSP. Support for the involvement of HSP in autoimmunity is provided by studies documenting enhanced autoantibody as well as cellular response to HSP 60/65 in several autoimmune diseases (Schoenfeld, Y et al Autoimmunity 2000 September; 15(2):199-202; U.S. Pat. No. 6,130,059 to Covacci, et al; and Gromadza G, et al Cerebrovascul Dis 2001, October; 12(3):235-39). The link between HSP 65 and atherosclerosis was initially recognized by George Wick's group, who found that normocholesterolemic rabbits immunized with different antigens developed atherosclerosis, provided the preparation used for immunization contained complete Freund's adjuvant (CFA)(Xu, Q, et al Arterioscler Thromb 1992;12:789-99). Since the major constituent of CFA is heat killed mycobacterium tuberculosis, the principal component of which is the HSP-65, they reasoned that the immune response towards this component led to the development of atherosclerosis. This was confirmed when these authors demonstrated that immunization of animals with HSP 65 produced pronounced atherosclerosis, and that T cells from experimentally atherosclerotic rabbits overexpressed HSP-65, indicating a localized immune reaction restricted to the stressed arterial vessel. The importance of endogenous HSP-65 in atherogenesis was further demonstrated by the acceleration of fatty streak formation following HSP-65 (or Mycobacterium tuberculosis) immunization of naïve mice (George J, et al Arterioscler Thromb Vasc Biol 1999; 19:505-10;).
Involvement of humoral immune mechanisms in response to HSP-65 were observed in atherosclerosis: a marked correlation has been found between high levels of anti-HSP65 antibodies and the extent of sonographically estimated carotid narrowing in a screen of healthy individuals (Xu Q. et al Lancet 1993; 341: 255-9; Xu Q. et al Circulation 1999; 100(11): 1169-74). In addition, in-vitro experiments with cultured endothelial cells have demonstrated the concentration and time dependent induction of endothelial cell adhesion to monocytes and granulocytes following incubation with HSP65.
The association of HSP 65 with atherogenesis has led to a number of proposed therapeutic applications. Observing that immune reactivity to HSP 65 correlated with both microbial (e.g. H. pylori) infection and atherosclerosis, Bernie et al (Eur Heart J 1998; 19:366-7) proposed antibiotic therapy for reduction of infection and anti-HSP antibodies. Similarly, Covacci, et al (U.S. Pat. No. 6,130,059) disclosed the use of H. pylori HSPs, and related peptides, for diagnostic and therapeutic applications in atherosclerosis.
Atherosclerosis and Autoimmune Disease
Because of the presumed role of the excessive inflammatory-fibroproliferative response in atherosclerosis and ischemia, a growing number of researchers have attempted to define an autoimmune component of vascular injury. In autoimmune diseases the immune system recognizes and attacks normally non-antigenic body components (autoantigens), in addition to attacking invading foreign antigens. The autoimmune diseases are classified as auto-(or self-) antibody mediated or cell mediated diseases. Typical autoantibody mediated autoimmune diseases are myasthenia gravis and idiopathic thrombocytopenic purpura (ITP), while typical cell mediated diseases are Hashimoto's thyroiditis and type I (Juvenile) Diabetes.
The recognition that immune mediated processes prevail within atherosclerotic lesions stems from the consistent observation of lymphocytes and macrophages in the earliest stages, namely the fatty streaks. These lymphocytes, which include a predominant population of CD4+ cells (the remainder being CD8+ cells) were found to be more abundant than macrophages in early lesions, as compared with the more advanced lesions, in which this ratio tends to reverse. These findings posed questions as to whether they reflect a primary immune sensitization to a possible antigen or alternatively, result from previously induced local tissue damage. Regardless of the factors responsible for the recruitment of these inflammatory cells to the early plaque, they seem to exhibit an activated state manifested by concomitant expression of MHC class II HLA-DR and interleukin (IL) receptor as well as leukocyte common antigen (CD45R0) and the very late antigen 1 (VLA-1) integrin. Thus, the inflammatory reaction of the early stages of the atherosclerotic lesion may be either the primary initiating event leading to the production of various cytokines by the local cells (i.e endothelial cells, macrophages, smooth muscle cells and inflammatory cells), or one form of the immune system's response to the hazardous process. Some of the cytokines which have been shown to be upregulated by the resident cells include TNF-α, IL-1, IL-2, IL-6, IL-8, IFN-γ and monocyte chemoattractant peptide-1 (MCP-1). Platelet derived growth factor (PDGF) and insulin-like growth factor (ILGF) which are expressed by all cellular constituents within atherosclerotic plaques have also been shown to be overexpressed, thus possibly intensifying the preexisting inflammatory reaction by a co-stimulatory support in the form of a mitogenic and chemotactic factor. Recently, Uyemura et al. (J Clin Invest 1996; 97: 2130-2138) have elucidated type 1 T-cell cytokine pattern in human atherosclerotic lesions exemplified by a strong expression of IFN-γ but not IL-4 mRNA in comparison with normal arteries. Furthermore, IL-12—a T-cell growth factor produced primarily by activated monocytes and a selective inducer of Th1 cytokine pattern, was found to be overexpressed within lesions as manifested by the abundance of its major heterodimer form p70 and p40 (its dominant inducible protein) mRNA.
Similar to the strong evidence for the dominance of the cellular immune system within the atherosclerotic plaque, there is also ample data supporting the involvement of the local humoral immune system. Thus, deposition of immunoglobulins and complement components has been shown in the plaques in addition to the enhanced expression of the C3b and C3Bi receptors in resident macrophages.
Valuable clues with regard to the contribution of immune mediated inflammation to the progression of atherosclerosis come from animal models. Immunocompromised mice (class I MHC deficient) tend to develop accelerated atherosclerosis as compared with immune competent mice. Additionally, treatment of C57BL/6 mice (Emeson E E and Shen M L Am J Pathol 1993; 142: 1906-1915) and New-Zealand White rabbits (Roselaar S E, et al J Clin Invest 1995; 96: 1389-1394) with cyclosporin A, a potent suppressor of IL-2 transcription resulted in a significantly enhanced atherosclerosis under “normal” lipoprotein “burden”. These latter studies may provide insight into the possible roles of the immune system in counteracting the self-perpetuating inflammatory process within the atherosclerotic plaque.
Atherosclerosis is not a classical autoimmune disease, although some of its manifestations such as the production of the plaque that obstructs the vasculature may be related to aberrant immune responsiveness. In classical autoimmune disease, one can often define very clearly the sensitizing autoantigen attacked by the immune system and the component(s) of the immune system which recognize the autoantigen (humoral, i.e. autoantibody or cellular, i.e. lymphocytes). Above all, one can show that by passive transfer of these components of the immune system the disease can be induced in healthy animals, or in the case of humans the disease may be transferred from a sick pregnant mother to her offspring. Many of the above are not prevailing in atherosclerosis. In addition, the disease definitely has common risk factors such as hypertension, diabetes, lack of physical activity, smoking and others, the disease affects elderly people and has a different genetic preponderance than in classical autoimmune diseases.
Treatment of inflammatory disease may be directed towards suppression or reversal of general and/or disease-specific immune reactivity. Thus Aiello, for example (U.S. Pat. Nos. 6,034,102 and 6,114,395) discloses the use of estrogen-like compounds for treatment and prevention of atherosclerosis and atherosclerotic lesion progression by inhibition of inflammatory cell recruitment. Similarly, Medford et al (U.S. Pat. No. 5,846,959) disclose methods for the prevention of formation of oxidized PUFA, for treatment of cardiovascular and non-cardiovascular inflammatory diseases mediated by the cellular adhesion molecule VCAM-1. Furthermore, Falb (U.S. Pat. No. 6,156,500) designates a number of cell signaling and adhesion molecules abundant in atherosclerotic plaque and disease as potential targets of anti-inflammatory therapies.
Since oxidized LDL, Beta2GPI and HSP 65 have been clearly implicated in the pathogenesis of atherosclerosis (see above), the contribution of these prominent plaque components to autoimmunity in atheromatous disease processes has been investigated.
Immune Responsiveness to Plaque Associated Molecules
It is known that Ox LDL is chemotactic for T-cells and monocytes. Ox LDL and its byproducts are also known to induce the expression of factors such as monocyte chemotactic factor 1, secretion of colony stimulating factor and platelet activating properties, all of which are potent growth stimulants. The active involvement of the cellular immune response in atherosclerosis has recently been substantiated (Stemme S, et al, Proc Natl Acad Sci USA 1995; 92: 3893-97), who isolated CD4+ within plaques clones responding to Ox LDL as stimuli. The clones corresponding to Ox LDL (4 out of 27) produced principally interferon-γ rather than IL-4. It remains to be seen whether the above T-cell clones represent mere contact with the cellular immune system with the inciting strong immunogen (Ox LDL) or that this reaction provides means of combating the apparently indolent atherosclerotic process.
The data regarding the involvement of the humoral mechanisms and their meaning are much more controversial. One recent study reported increased levels of antibodies against MDA-LDL, a metabolite of LDL oxidation, in women suffering from heart disease and/or diabetes (Dotevall, et al., Clin Sci 2001 November; 101(5): 523-31). Other investigators have demonstrated antibodies recognizing multiple epitopes on the oxidized LDL, representing immune reactivity to the lipid and apolipoprotein components (Steinerova A, et al., Physiol Res 2001;50(2): 131-41) in atherosclerosis and other diseases, such as diabetes, renovascular syndrome, uremia, rheumatic fever and lupus erythematosus. Several reports have associated increased levels of antibodies to Ox LDL with the progression of atherosclerosis (expressed by the degree of carotid stenosis, severity of peripheral vascular disease etc.). Most recently, Sherer et al (Cardiology 2001;95(1):20-4) demonstrated elevated levels of antibodies to cardiolipin, β-2GPI and oxLDL, but not phosphatidylcholine or endothelial cells in coronary heart disease. Thus, there seems to be a consensus as to the presence of anti-plaque-component antibodies in the form of immune complexes within atherosclerotic plaque.
Antibodies to Ox LDL have been implicated in both normal and pathological lipoprotein metabolism. Thus, it is known that immune complexes of Ox LDL and its corresponding antibodies are taken up more efficiently by macrophages in suspension as compared with Ox LDL. No conclusions can be drawn from this consistent finding on the pathogenesis of atherosclerosis since the question of whether the accelerated uptake of Ox LDL by the macrophages is beneficial or deleterious has not yet been resolved.
Important data as to the significance of the humoral immune system in atherogenesis comes from animal models: hyperimmunization of LDL-receptor deficient rabbits with homologous oxidized LDL, resulted in the production of high levels of anti-Ox LDL antibodies and was associated with a significant reduction in the extent of atherosclerotic lesions. Likewise, a decrease in plaque formation followed the immunization of rabbits with cholesterol rich liposomes and stimulation of production of anti-cholesterol antibodies; however, this effect was accompanied by a undesirable 35% reduction in very low density lipoprotein cholesterol levels.
Regarding the immunogenicity of Beta2GPI, it has been shown that Beta2GPI serves as a target antigen for an immune-mediated attack, influencing the progression of atherosclerosis in humans and mice. George J et al. immunized LDL-receptor deficient mice with Beta2GPI, producing a pronounced humoral immune response to human Beta2GPI, and larger early atherosclerotic lesions in comparison with controls (George J, et al Circulation 1998; 15:1108-15). Afek A, et al obtained similar results in atherosclerosis-prone apolipoprotein-E-knockout mice immunized once with human Beta2GPI and fed a high fat diet for 5 weeks (Afek A et al. Pathobiology 1999;67:19-25).
Further, although immune reactivity to Beta2GPI in humans with the prothrombotic antiphospholipid syndrome has traditionally been attributed to the presence of autoantibodies to Beta2GPI, recent observations have indicated the importance of a cellular immune response to Beta2GPI. T-cells reactive with Beta2GPI have been demonstrated in the peripheral blood of patients with antiphospholipid syndrome. These T cells displayed a T-helper-1 phenotype (secreting the proinflammatory (and proatherogenic) cytokine interferon-) and were also capable of inducing tissue factor production (Visvanathan S., and McNiel H P. J Immunolog 1999; 162:6919-25). Taken together, the abundant data gathered to date regarding anti Beta2GPI (for review see Roubey R A, Curr Opinion Rheumatol 2000; 12:374-378), indicates that the immune response to this plaque related antigen may play a significant role in influencing the size and composition of atherosclerotic plaque.
Finally, there exists a significant dependency in the antigenicity, and pathogenicity of oxidized phospholipids and Beta2GPI. As mentioned above, some of the autoimmune epitopes associated with minimally modified LDL and Beta2GPI are cryptic. Kyobashi, et al (J Lipid Res 2001; 42:697-709), and Koike, et al (Ann Med 2000; 32:Suppl I 27-31) have identified a macrophage-activating oxLDL specific ligand present only with Beta2GPI-OxLDL complex formation. This ligand was recognized by APLS-specific autoantibodies. Thus, both the pathogenic role of oxidized LDL and other plaque components, and their importance as autoantigens in atherosclerosis, as well as other diseases, have been extensively demonstrated in laboratory and clinical studies.
Mucosal Tolerance in Treatment of Autoimmune Disease
Recently, new methods and pharmaceutical formulations have been found that are useful for treating autoimmune diseases (and related T-cell mediated inflammatory disorders such as allograft rejection and retroviral-associated neurological disease). These treatments induce tolerance, orally or mucosally, e.g. by inhalation, using as tolerizers autoantigens, bystander antigens, or disease-suppressive fragments or analogs of autoantigens or bystander antigens. Such treatments are described, for example, in U.S. Pat. No. 5,935,577 to Weiner et al. Autoantigens and bystander antigens are defined below (for a general review of mucosal tolerance see Nagler-Anderson, C., Crit Rev Immunol 2000;20(2):103-20). Intravenous administration of autoantigens (and fragments thereof containing immunodominant epitopic regions of their molecules) has been found to induce immune suppression through a mechanism called clonal anergy. Clonal anergy causes deactivation of only immune attack T-cells specific to a particular antigen, the result being a significant reduction in the immune response to this antigen. Thus, the autoimmune response-promoting T-cells specific to an autoantigen, once anergized, no longer proliferate in response to that antigen. This reduction in proliferation also reduces the immune reactions responsible for autoimmune disease symptoms (such as neural tissue damage that is observed in multiple sclerosis; MS). There is also evidence that oral administration of autoantigens (or immunodominant fragments) in a single dose and in substantially larger amounts than those that trigger “active suppression” may also induce tolerance through anergy (or clonal deletion).
A method of treatment has also been disclosed that proceeds by active suppression. Active suppression functions via a different mechanism from that of clonal anergy. This method, discussed extensively in PCT Application PCT/US93/01705, involves oral or mucosal administration of antigens specific to the tissue under autoimmune attack. These are called “bystander antigens”. This treatment causes regulatory (suppressor) T-cells to be induced in the gut-associated lymphoid tissue (GALT), or bronchial associated lymphoid tissue (BALT), or most generally, mucosa associated lymphoid tissue (MALT) (MALT includes GALT and BALT). These regulatory cells are released in the blood or lymphatic tissue and then migrate to the organ or tissue afflicted by the autoimmune disease and suppress autoimmune attack of the afflicted organ or tissue. The T-cells elicited by the bystander antigen (which recognize at least one antigenic determinant of the bystander antigen used to elicit them) are targeted to the locus of autoimmune attack where they mediate the local release of certain immunomodulatory factors and cytokines, such as transforming growth factor beta (TGF beta), interleukin-4 (IL-4), and/or interleukin-10 (IL-10). Of these, TGF-beta is an antigen-nonspecific immunosuppressive factor in that it suppresses immune attack regardless of the antigen that triggers the attack. (However, because oral or mucosal tolerization with a bystander antigen only causes the release of TGF-beta in the vicinity of autoimmune attack, no systemic immunosuppression ensues.) IL-4 and IL-10 are also antigen-nonspecific immunoregulatory cytokines. IL-4 in particular enhances (T helper 2) Th2 response, i.e., acts on T-cell precursors and causes them to differentiate preferentially into Th2 cells at the expense of Th1 responses. IL-4 also indirectly inhibits Th1 exacerbation. IL-10 is a direct inhibitor of Th1 responses. After orally tolerizing mammals afflicted with autoimmune disease conditions with bystander antigens, increased levels of TGF-beta, IL-4 and IL-10 are observed at the locus of autoimmune attack (Chen, Y. et al., Science, 265:1237-1240, 1994). The bystander suppression mechanism has been confirmed by von Herreth et al., (J. Clin. Invest., 96:1324-1331, September 1996).
More recently, oral tolerance has been effectively applied in treatment of animal models of inflammatory bowel disease by feeding probiotic bacteria (Dunne, C, et al., Antonie Van Leeuwenhoek 1999 Jul-Nov; 76(1-4):279-92), autoimmune glomerulonephritis by feeding glomerular basement membrane (Reynolds, J. et al., J Am Soc Nephrol 2001 January; 12(1): 61-70) experimental allergic encephalomyelitis (EAE, which is the equivalent of multiple sclerosis or MS), by feeding myelin basic protein (MBP), adjuvant arthritis and collagen arthritis, by feeding a subject with collagen and HSP-65, respectively. A Boston based company called Autoimmune has carried out several human experiments for preventing diabetes, multiple sclerosis, rheumatoid arthritis and uveitis. The results of the clinical trials have been less impressive than the animal experiments, however there has been some success with the prevention of arthritis.
Oral tolerance to autoantigens found in atherosclerotic plaque lesions has also been investigated. Study of the epitopes recognized by T-cells and Ig titers in clinical and experimental models of atherosclerosis indicated three candidate antigens for suppression of inflammation in atheromatous lesions: oxidized LDL, the stress-related heat shock protein HSP 65 and the cardiolipin binding protein beta 2GP 1. U.S. patent application Ser. No. 09/806,400 to Shoenfeld et al (filed Sep. 30, 1999), which is incorporated herein in its entirety, discloses the reduction by approximately 30% of atherogenesis in the arteries of genetically susceptible LDL receptor deficient mice (LDL-RD) fed oxidized human LDL. Although significant inhibition of atherogenesis was achieved, presumably via oral tolerance, no identification of specific lipid antigens or immunogenic LDL components was made. Another obstacle encountered was the inherent instability of the orally fed antigen in vivo, due to digestive breakdown, and uptake of oxidized LDL by the liver and cellular immune mechanisms. It is plausible that a mucosal route of administration other than feeding (oral) would have provided tolerance of greater efficiency.
The induction of immune tolerance and subsequent prevention or inhibition of autoimmune inflammatory processes has been demonstrated using exposure to suppressive antigens via mucosal sites other than the gut. The membranous tissue around the eyes, the middle ear, the respiratory and other mucosa, and especially the mucosa of the nasal cavity, like the gut, are exposed to many invading as well as self-antigens and possess mechanisms for immune reactivity. Thus, Rossi, et al (Scand J Immunol 1999 August; 50(2):177-82) found that nasal administration of gliadin was as effective as intravenous administration in downregulating the immune response to the antigen in a mouse model of celiac disease. Similarly, nasal exposure to acetylcholine receptor antigen was more effective than oral exposure in delaying and reducing muscle weakness and specific lymphocyte proliferation in a mouse model of myasthenia gravis (Shi, FD. et al, J Immunol 1999 May 15; 162 (10): 5757-63). Therefore, immunogenic compounds intended for mucosal as well as intravenous or intraperitoneal administration should be adaptable to nasal and other membranous routes of administration.
Thus, there is clearly a need for novel methods of employing, and compositions of plaque associated molecules capable of superior tolerizing immunogenicity in mucosal, especially nasal, administration.